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Synthesis and characterization of site-selective orbitide-BSA conjugate to produce antibodies Pramodkumar D Jadhav, Youn Young Shim, and Martin J.T. Reaney Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00357 • Publication Date (Web): 14 Sep 2016 Downloaded from http://pubs.acs.org on September 15, 2016

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Bioconjugate Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Bioconjugate Chemistry

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Synthesis and characterization of site-selective orbitide-

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BSA conjugate to produce antibodies

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Pramodkumar D. Jadhav,† Youn Young Shim,*,†,‡ and Martin J. T. Reaney*,†,‡,§

5 6

†

7

Saskatchewan S7N 5A8, Canada

8

‡

9

§

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Department of Plant Sciences, University of Saskatchewan, 51 Campus Drive, Saskatoon,

Prairie Tide Chemicals Inc., 102 Melville Street, Saskatoon, Saskatchewan S7J 0R1, Canada

Guangdong Saskatchewan Oilseed Joint Laboratory, Department of Food Science and Engineering,

Jinan University, 601 Huangpu Avenue West, Guangzhou, Guangdong 510632, China

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AUTHOR INFORMATION

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Email address: (P.D.J.) [email protected]; (Y.Y.S.) [email protected]; (M.J.T.R.)

25

[email protected].

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ABSTRACT

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Bioactive flax cyclic peptides (orbitides, linusorbs) were site specifically ligated through methionine

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with bovine serum albumin (BSA) to produce immunogenic compounds. In this study, modified

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flaxseed immunosuppressant orbitides (linusorbs, LOs) containing hydroxyl (OH) groups were

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synthesized for use as haptens. These compounds were extensively characterized by 1H-NMR,

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NMR, HPLC-MS/MS, and FTIR. The haptens were conjugated to BSA and the extent of hapten

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incorporation was determined by MALDI, LC-ESI-MS, and SDS-PAGE. The BSA hapten complexes

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were used to elicit polyclonal antibody (pAbs) production in rabbits. A competitive indirect enzyme-

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linked immunosorbent assay (CI-ELISA) was developed that used orbitide-specific pAbs and

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horseradish peroxidase (HRP) conjugates. The LO assay detection limit was approximately 0.01 µg/mL

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(ppm) and, thus, ELISA can be used for the detection of LOs in tissue and plant samples. The pAbs, the

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first formed against an Ab hapten, can be used to detect and quantify LOs in flax and flaxseed samples,

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to verify the presence of LOs in flaxseed containing foods, and detection of LOs in tissue samples,

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wastes and body fluids of animals fed flaxseed.

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Keywords: polyclonal linusorb antibody; ELISA; conjugate of linusorb and BSA; orbitide;

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cyclolinopeptide; flaxseed; Linum usitatissimum L.

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C-

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INTRODUCTION

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Therapeutic peptides have gained importance as potential drug candidates for applications including

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anti-microbial therapy, anti-cancer vaccines, Alzheimer’s therapy, and malaria prophylaxis.1-4 Hence it

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is important to monitor drug stability in vivo and antibody assays are useful tools for this application.5

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Therapeutic peptides include cyclic hydrophobic peptides such as the immunosuppressant cyclosporine

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A (CsA) and the growth inhibiting hormone somatostatin. These have been used to induce specific

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polyclonal antibodies (pAbs). The pAbs of CsA were used to monitor the cyclosporine levels using

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radioimmunoassay and somatostatin pAbs were used to detect somastotatin receptors in carcinoid

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tumors.6,7

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Linusorb (LO) 1, a natural cyclic nonapeptide, was first isolated in 1959 from flaxseed (Linum

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usitatissimum L.).8 Twenty eight LOs containing eight to ten amino acids have since been isolated from

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flaxseed.9-12 Structures of tryptophan and glycine free LOs are shown in Table 1 as examples that are

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pertinent to this study.

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Table 1. Amino Acid Sequences and Chemical Formulas of flaxseed LO code LOa 1

[1–9-NαC]-linusorb B3

2

[1–9-NαC]-linusorb B2

3 4 5 6 7

58 59 60

[1–9-NαC],[1-(Rs,Ss)MetO]-linusorb B2 [1–9-NαC],[1-(Rs,Ss)MetO2]-linusorb B2 [1–8-NαC]-linusorb B1 [1–8-NαC],[1-(Rs,Ss)MetO]-linusorb B1 [1–8-NαC],[1-(Rs,Ss)MetO2]-linusorb B1

prior amino acid sequence (NαC-)b designation Ile-Leu-Val-Pro-Pro-Phe-PheCLA Leu-Ile Met-Leu-Ile-Pro-Pro-Phe-PheCLB Val-Ile MetO-Leu-Ile-Pro-Pro-Phe-PheCLC Val-Ile MetO2-Leu-Ile-Pro-Pro-Phe-PheCLK Val-Ile Met-Leu-Val-Phe-Pro-Leu-PheCLE' Ile MetO-Leu-Val-Phe-Pro-LeuCLE Phe-Ile MetO2-Leu-Val-Phe-Pro-LeuCLJ Phe-Ile

chemical formula

protonated ion mass (m/z)c

C57H85N9O9

1040.66

C56H83N9O9S

1058.61

C56H83N9O10S

1074.60

C56H83N9O11S

1090.60

C51H76N8O8S

961.57

C51H76N8O9S

977.56

C51H76N8O10S

993.56

a

Linusorb naming based on the proposed nomenclature of orbitides.46 b Abbreviations are MetO for methionine S-oxide, and MetO2 for methionine S,S-dioxide. c From reference and ESI-MS data.11 3 ACS Paragon Plus Environment

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Methionine containing LOs (2 and 5) were also present in oxidized form as methionine sulfoxide

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LOs (3 and 6) and methionine sulfone LOs (4 and 7) respectively. In addition, LOs 4 and 7 were

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chemically synthesized from their parent peptides LOs 3 and 6, respectively but they can also be

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obtained from oxidized flaxseed oil.13,14 LOs 1, 2, 3 and 6 have shown cytotoxic activity against various

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cancer cell lines.15 1 was found to possess immunosuppressive activity comparable to that of CsA.16 The

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strong immunosuppressive activity of 1 makes the peptide very interesting from the point of view of its

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effects when consumed in food and possible utilization in medicine.16 Of all the 28 LOs, 1 showed the

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best immunosuppressive activity (IC50-2.5 μg mL-1) followed by 4 and 7.17 The molecular mechanism

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of 1 and CsA is similar, i.e. they complex with cyclophilin, interact with the calcineurin system and

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inhibit its phosphatase activity.18 It has been reported that binding sites (DAL8-MVA11) of CsA with

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cyclophilin A are similar to the antibody raised against it.19 Similarly in 1, the sequence responsible for

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binding to cyclophilin A is reported to be Val-Pro-Pro-Phe20 and hence the antibody raised against 1

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might also have the same binding specificity as cyclophilin.

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Peptide antibodies are important tools for purification, protein identification, diagnostic, and

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therapeutic purposes. Peptide modification through organic synthesis helps to generate varied sequences

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of amino acids and generate application specific antibodies. Peptides are conjugated with carrier

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proteins like ovalbumin, bovine serum albumin (BSA), and keyhole limpet hemocyanin to elicit

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antibodies.21-23 Commonly a functional group is chosen for covalent coupling based on a facile and non-

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destructive chemical approach. The ε-amino group of lysine or the thiol group of cysteine are often

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chosen for conjugation through a one-pot two-step synthetic method.24 The most common ways to

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produce pAbs through peptide conjugation are by conjugating to carrier proteins or by coupling with

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lysine on a multivalent core (multiple antigenic peptides).25 BSA is often chosen as it contains about 59

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lysine amino residues for conjugation. It is highly soluble and has a moderate molecular weight (66

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kDa).26 In addition, high purity BSA is readily available. Anti-peptide antibodies are useful in analytical

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applications including immunoprecipitation, immunoaffinity chromatography, Western blotting analysis 4 ACS Paragon Plus Environment


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and enzyme-linked immunosorbent assay (ELISA).5,27-29 Anti-peptide pAbs have been used to detect the sexually transmitted pathogen, Mycoplasma genitalium through immunofluorescence staining.29 pAbs are also used as immunoblots, an analytical tool to determine the native or denatured calreticulin in solution.30 This paper describes generation of pAbs against 4 and 7, where 4 is an analog of 1. These pAbs might be used in two ways: therapeutic drug monitoring (localization) of peptides and detection of LOs in flax and flaxseed products. For example, antibodies might be used as tools for detection of LOs to investigate their in vivo stability and pharmacokinetics. In addition, they might also be used to detect LOs in flaxseed samples. The total concentration of LOs is only 0.2% of seed mass and several analytical protocols have been developed for measuring these compounds.9,10,31,32 However, not all analytical methods are satisfactory for LO detection in multi-component mixtures such as serum or plant roots. In contrast, an ELISA provides highly sensitive, rapid, and precise means to estimate biological parameters, with the added advantage that it can rapidly analyze a large number of samples. ELISA uses enzyme and antibody such that quantification of nanograms of analyte is possible. 4 and 7 are the oxidized products of their precursor orbitides 3 and 6, are not present in freshly ground flaxseed and are indicators of aging flaxseed.13 The preparation of antibodies from these LOs will help to develop an ELISA protocol that can be used for LO identification and quantitation in flaxseed. It was reported that LO 6 is responsible for the bitter flavor of flaxseed oil.31 Since these generated pAbs will have affinity to LO 6, an ELISA might be able to detect LO 6 (bitter flavor) in oil. ELISA assays can be used for both therapeutic drug monitoring and detection in flaxseed samples. In addition to the possible use of LOs as drugs these substances occur in food, animal feed and botanical materials. Flaxseed, ground flaxseed, flaxseed oil and partially defatted flaxseed meal are food and animal feed materials that contain LOs. An ELISA could prove valuable in monitoring LOs in these materials and in tissues of subjects consuming them, and other, flaxseed products. Additionally, tissues of flax plants, particularly flax roots also contain LOs. 5 ACS Paragon Plus Environment

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RESULTS AND DISCUSSION In this study, we have selected two types of LOs, 4 and 7 containing nine and eight amino acid residues. However, 4 and 7 contain a methionine S, S-dioxide moiety in their side chain and they are oxidised product of LOs (2 and 3) and LOs (5 and 6) with the same fundamental parent peptides 2 and 5. We have devised methods to chemically modify methionine in the parent LOs 2 and 5 in a systematic fashion, without changing other amino acids.33 Here we report synthesis and full characterization of novel LO analogs with hydroxyl side chain to be used as the point of attachment to a carrier protein. The hydroxyl of LOs were activated by reacting with carbonyldiimidazole and then coupled with the carrier protein (BSA). The number of LOs (haptens) attached to BSA was characterized and BSA conjugated peptides were used to produce antibodies. Synthesis of Haptens. The sulfone ester LOs 9 and 13 were synthesized as described previously (Figure S1).33 These esters have to be modified to obtain more reactive functional groups that can be coupled with the carrier protein. This can be performed in two ways, first hydrolyzing the ester to acid and prepare succinimidyl ester or to attach activated hydroxyl group along the side chain. The latter process was useful because we can use ethanolamine to react with sulfone ester and introduce a four atom linker/spacer. This linker will help expose the hapten on the carrier protein surface and allow better recognition by the immune system. In addition, the sulfone ester-linker was situated away from the bioactive fragment present in LOs 9 and 13 and might avoid shielding of the hapten by the carrier protein. These esters were treated with ethanolamine in methanol under reflux conditions to obtain hydroxyl side chain containing LOs 10 and 14 (Figure S2). The course of the reaction was followed via analytical HPLC and when the starting material was consumed, methanol was evaporated to obtain the crude product. These crude LOs were purified using preparative HPLC and were isolated in good yield. Optimization of Coupling Reaction of Peptide with 1,1'-carbonyldiimidazole. The primary hydroxyl groups of the corresponding peptides were activated using 1,1'-carbonyldiimidazole (CDI) to form active imidazoyl carbamate. It was observed that hapten is insoluble in water while BSA 6 ACS Paragon Plus Environment


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is water soluble. Hence activation of LOs should be performed in a non-aqueous but water miscible solvent such as DMSO, DMF and THF. This allows both activation and conjugation of hapten with BSA. The reaction conditions were optimized using different conditions including solvents, reaction times, equivalence ratios (1:2 to 1:4), and temperatures. When 10 was reacted with CDI (2 eq) in DMSO at room temperature, it yields only starting material and a side product of molecular mass 1148.6 (Figure 1A). In addition, by changing the solvent to THF and refluxing the reaction also gives a mixture of products (data not shown). However, when the reaction was performed in THF at room temperature with CDI (4 eq) a peptide-CDI adduct with a molar mass of 1235.6 (Figure 1B) was formed. But before reacting with BSA, we reacted this with the primary amine (butyl amine) to verify its reactivity. This reaction resulted in the formation of the expected peptide-butyl amine complex of molar mass 1276.7 (Figure 1C).

A

B


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C

Figure 1. CID MS spectra of 9 with CDI (A), with THF (B), and reaction with BSA (C) showing fragmentation pattern. Hapten Conjugation. The peptides are small molecules and might produce a weak immune response, hence we coupled them with carrier proteins to produce high titer Ab.25 The LOs were coupled with carrier proteins such as BSA for Ab production using 10 and 14 as haptens in immunogen preparation and competitors in ELISA. These molecules can easily couple with a carrier protein like BSA using reactive functional groups (–OH group) present on modified side chains (Scheme 1).

Scheme 1. Synthesis and conjugation of hapten with protein. 8 ACS Paragon Plus Environment


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To obtain high antibody titer it is necessary to attain a mole ratio for conjugated hapten to BSA of greater than 10:1. LOs containing –OH group were activated by reacting with CDI and finally coupled with BSA through –NH2 residues to form stable covalent amide bonds. LOs contain hydrophobic amino acids in their structure and have low aqueous solubility. Hence LO-CDI intermediate was reacted with BSA at higher pH (borate buffer, pH-8.5). The CDI intermediate was reacted with BSA in borate buffer in different ratios (Table 2). Table 2. Different Molar Ratios of BSA-Hapten and Their MALDI BSA:hapten ratio

11 conjugate

15 conjugate

1:30

66752

67458, 68564

1:120

66221

66298

1:480

66251, 69081, 71732

NDa

1:51

89463, 77871, 66989

90357, 89857, 78409, 68172

a

ND: not detected.

When the reaction was conducted at +4 °C, THF clathrates formed with water in the sample. Hence the reaction was performed at room temperature, but it was observed that the CDI intermediate was experiencing hydrolysis in water and there was less hapten incorporation for both 10 and 14. In addition, increasing the ratio of hapten to BSA (30:1; 120:1; 480:1) also has less impact on incorporation (Table 2). To overcome the hydrolysis rate of CDI intermediate and increase nucleophilic substitution, we used a multiple addition strategy by adding CDI intermediate over different time intervals. This procedure increased the amount of hapten bound per BSA protein. The unbound haptens were removed from BSA conjugates by extensive dialysis in aqueous buffer before freeze drying the reaction product. A 1:51 ratio of BSA:hapten were further characterized using ESI-QTOF, MALDI, and SDS-PAGE and used to produce pAbs. Characterization of Hapten and Conjugates. Structure of Hapten. The structures of 10 and 14 were confirmed by MS/MS and NMR methods. HPLC-MS analysis showed a quasimolecular ion peak at m/z 1177.6730 [M+H]+ corresponding to a molecular formula, C59H89N10O13S of 10 and m/z 1080.5921 [M+H]+ corresponding to a molecular 9 ACS Paragon Plus Environment

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formula, C54H82N9O12S of 14. MS/MS fragmentation pattern shows the amino acid sequence of both these derivatives (Figures S3A and S3B) and also confirms the attachment of hydroxyl side chain along the MetO2 residue. The IR absorption peaks at 3329 cm-1 and 1665 cm-1, characteristic of all known LOs, confirmed the presence of amide linkages in 10 and 14, whereas, those at 1320 and 1122 cm-1 were attributed to the presence of a MetO2 residue and broad band at 3334 shows presence of hydroxyl groups in 10 and 14.9,10,13 The CID MS/MS and NMR spectral data of 10 (Figure S3A and experimental section) showed remarkable similarities to that of 4.13 However, a fragment loss of m/z 250 in the MS/MS data of 10 (Figure S3A) in place of the loss of m/z 163 (MetO2 in 4) confirms the attachment of an alcohol side chain to the MetO2 residue in 10. The α protons were assigned from 1H-NMR (Figures S4 and S5) and sequential assignment of β, γ and δ protons were done using 1H-1H corelation spectroscopy (COSY) (Figures S6 and S7) and elucidation of attachments of these protons to carbon atoms were performed by heteronuclear multiple quantum coherence (HMQC) (Figures S8 and S9). In addition, coupling to amide protons and carbonyl carbons were determined using nuclear overhauser effect (NOE) (Figures S10 and S11) and heteronuclear multiple bond coherence (HMBC) correlations (Figures S12 and S13). The selected NOE and HMBC correlations were also shown in Figure 2.



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Figure 2. Structure of 10 (A) and 14 (B). Double arrows show selected NOE correlations. Half arrows show selected HMBC correlations. These spectra were used to confirm the orbitide amino acid sequence. The structure of 10 and its parent peptide 4 exist as single isomer. However 14 shows the presence of only one isomer as compare to the parent peptide 7 containing two isomers. This was shown in the 13C-NMR spectrum of 7 with two sets of signals for each carbon atom and 1H-NMR displayed two sharp singlets at δ 2.86 and 3.00 ppm corresponded to MetO2 moieties of the two conformers.13 However 14 showed only one set of sharp 13

C-NMR signals (Figure S14). These observations suggested that 14 might exist as single stable

conformer under the NMR experimental conditions. The hydroxyl group in the side chain of 14 might form hydrogen bonds that stabilize the molecule in one conformation. MS/MS profile of 14 also shows attachment of the alcohol side chain. 1D and 2D NMR spectra of 10 and 14 are provided in the supporting documents (Figures S4–S15). Following the preparation of modified orbitides 10 and 14 it was important to investigate the occurrence of structural changes in the orbitide core structure. NMR spectroscopy was used to compare 11 ACS Paragon Plus Environment

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the structural signals of the starting material 4 and 7 to those of the conjugate products 10 and 14, respectively. It was reported that chemical shifts of α protons of amino acids can be used to determine the extent, identity and location of secondary structural elements present in proteins.34 This approach, while informative, is not a substitute for two- and three-dimensional NMR structural analysis, but it can provide complimentary information.34-36 Some of the examples for use of α protons of each amino acid residue were studied to determine conformational changes in Kalata B1 and α-Conotoxin ImI.37,38 Our observation from chemical shift analyses showed that most amino acid residues (six out of nine amino acid residues for 10 show no significant perturbations in NMR resonances when compared to its’ parent compound 4. This indicates that the substitution on the MetO2 side chain did not impact orbitide conformation. However significant perturbations were seen for 14 when compared to 7, hence the conformation of parent and modified compound are likely significantly different (Table 3). Tables 3. Chemical Shift Differences of αH Signals for 10 and 14 Compared with the 4 and 7, Respectively αH assignment MetO21 2

Leu IIe

3

Pro4 5

Pro

4

10

4.81

4.76

4.07

4.08

αH assignment MetO21

4.76

4.88

4.36

4.00

3

4.50

4.20

Leu

4.52

4.45

Val

3.90

3.71

Phe4

4.34

4.79

4.08

5

4.09

4.48

6

3.81

3.45

4.96

5.33

4.13

4.07

4.07

Pro

4.11

4.84

Leu

Phe7

4.96

3.98

Phe7

Val IIe

8

14

2

6

Phe

7

3.90

3.86

Ile

8

9

4.60 4.51 This might occur due to hydrogen bonding of the hydroxyl group. We have previously reported that 7

is present in two isomeric forms.13 In contrast, 10 and 14 were present as single isomers. The stability of the single isomer may be due to hydrogen bonding of the hydroxyl side chain. Chemical shift analysis showed that there might be structural changes induced by the alcohol side chain in 10, but there are major structural changes in 14 compare to the parent LOs. After coupling with BSA this molecule will



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no longer participate in this form of hydrogen bond formation and it might have a conformation that is more similar to its parent compound than the intermediate. The characterization of protein conjugates was important to determine the hapten density as this aids in the production of higher antibody titer. Several techniques such as gel electrophoresis, UV-difference spectroscopy and TNBS titration have been used. However, the mass spectrometric techniques MALDI and ESI-MS provide the sensitivity and accuracy required for determining hapten density. In this study, we have used SDS-PAGE, MALDI and ESI-MS to characterize conjugates of 11 and 15. SDS-PAGE. Hapten incorporation by forming covalent bounds to a protein increases the molecular mass and possibly decreases its SDS-PAGE mobility. After conjugation, bands attributed to the conjugate of LO with BSA were broader and less mobile than unreacted BSA (66 kDa) (Figure 3).

Figure 3. SDS-PAGE patterns of 11 (A) and 15 (B) conjugates that were used for production of anti-11 and anti-15 pAbs. This result is consistent with the formation of BSA covalently bound to LO. The LO-BSA conjugates shown smeared bands in gel electrophoresis. This prevents determination of exact molecular mass(es) of the LO-BSA conjugates.26 SDS-PAGE mass discrimination (

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